Progress 09/01/06 to 08/31/08
Outputs
OUTPUTS: The Silybum marianum flavonolignans silychristin, silydianin, silibinin, and isosilibinin were extracted in triplicate using pressurized hot water extraction (PHWE) with a Parr extraction vessel. The four flavonolignans were identified by LC/MS, LC/MS/MS and exact mass determination, both before and after pressurized hot water extraction. Each flavonolignan was evaluated for its athero-protective effect both before and after extraction to assess the contribution of the byproducts formed during extraction. Endpoints used to evaluate the athero-protective effects of the extracts were the amount of oxidation to low-density lipoprotein (LDL), as measured by the thiobarbituric acid reacting-substances (TBARS) assay, and the formation and attenuation of Reactive Oxygen Species (ROS) in RAW 264.7 macrophage cells. Flavonolignans were then extracted using the aforementioned Parr reactor (bench scale), an ethanol Soxhlet apparatus, and a pilot scale apparatus designed for PHWE. Silymarin yields and degradation profiles from each type of extraction were compared. The results have been or are being disseminated to communities of interest through the publication process and by presentation. Results were presented at the 30th Symposium on Biotechnology for Fuels and Chemicals. Results were also presented via poster format at the National Research Initiative Project Director's Meeting for Improving Food Quality and Value. Activities during which the project information were disseminated include an undergraduate Chemical Engineering Design II class, under the direction of Dr. Roy Penney, where students helped in the design of the pilot scale extractor, and training of a Biological and Agricultural Engineering undergraduate student, Kris Bunnell, who will use the extractor for his senior honors thesis project. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Silychristin, silydianin, silibinin and isosilibinin in 2 mL methanol added to 198 mL water were exposed to pressurized hot water (PHW) at 500 kPa and 413 K for 0.5 hr at 150 rpm, then were dried down by SpeedVac and reconstituted in methanol for HPLC-UV and LC/MS/MS analyses. Aliquots were diluted in DMSO for a dose response for assays. Oxidation was determined by TBARS measurement and ROS attenuation. Degradation peaks and standards were analyzed by HPLC-UV and LC-MS/MS. A degradation product in silychristin had a m/z of 479, not 481, and did not show any fragmentation in the m/z 325-355 range. All flavonolignans formed degradation products after PHWE. Silybinin was athero-protective at 75 uM before and after PHWE. Silychristin, before PHWE, was protective at a dose of 38 uM, while 150 uM was required for protection after PHWE. Silydianin was lowered TBARS formations at a high dose of 300 uM before PHWE; after extraction, with 7 degradation products, 38 uM had athero-protective effects. Isosilbinin afforded significant protection at 75 uM, while its extracted counterpart worked at 75 uM. Dose-dependent inhibitions in TBARS formations were observed in 0-300 uM silibinin, silychristin, silydianin, and isosilibinin both before and after PHWE. Inhibitions in TBARS formation paralleled those observed when using the same doses of silibinin, silychristin, silydianin, and isosilibinin exposed to PHW. Attenuation of ROS formation in RAW 264.7 macrophage cells was good with higher doses of all tested flavonolignans. Degradation products do not greatly diminish the athero-protective effects of the silychristin or silibinin, and in fact slightly improved the athero-protective capacities of silydianin and isosilibinin. Silymarin fruits were extracted with PHW using a Parr extractor (above), and similarly with a Soxhlet apparatus, first defatted for 1 hr using 200 mL petroleum ether, then extracted 4 hr with 200 mL ethanol. Extracts were dried down (above). The 2 methods' yields were compared to those from a bench scale apparatus designed for PHWE. 250 g fruits were fed in 25 g feed shots every 3 min over a 30 min extraction duration. 9.45 L water was counter-current fed over 30 min, with a flow rate of 0.315 L/min. A stainless steel auger maintained an agitation rate of 1.5 rpm. Pressure was held above atmospheric, at approximately 138 kPa. 1 mL samples were collected at 9, 18, and 24 min, evaporated under nitrogen, and reconstituted in 1 mL methanol for HPLC analyses. Total silymarin yields from the Parr reactor, Soxhlet apparatus, and bench scale PHWE, in mg silymarin per g of seed, were 0.23, 7.61, and 6.22, respectively. When using the bench scale extractor, silymarin yields increased over time. At 9, 18, and 24 min, silymarin yields reached 1.34, 10.71, and 24.94 mg per g of seed, respectively. These comparative extraction experiments show the potential of using PHW as an alternative solvent. Yields were inconsistent when using water versus ethanol, but fine tuning of the bench scale extractor could minimize these variations and render supercritical water as a promising extraction solvent for silymarin fruits.
Publications
- Wallace, S., Vaughn, K., Stewart, B., Viswanathan, T., Clausen, E., Nagarajan, S., and Carrier, D.J. 2008. Milk thistle extracts inhibit the oxidation of low-density lipoprotein and subsequent scavenger receptor-dependent monocyte adhesion. J. Agric. Food Chem. 56: 3966-3972.
|
Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: One of the major goals of the project was to generate, identify and quantify the typical thermal degradation products formed during the extraction of Silybum marianum fruits with subcritical water. The flavonolignans silichristin, silidianin, silibinin and isosilibinin from S. marianum fruits were exposed to subcritical water at 500 kPa and 413 K for 0.5 hr to generate thermal degradation products. Each experiment was performed in triplicate. The four flavonolignans and their degradation products were identified by LC/MS, LC/MS/MS and exact mass determination, both before and after subcritical water contact. A second major goal of the project was to evaluate each of the flavonolignans for their athero-protective effects both before and after subcritical water contact to assess the negative or positive contributions of the degradation products. The endpoints used to evaluate the athero-protective effects were the amount of oxidation to low-density lipoprotein (LDL), as
measured by the thiobarbituric acid reacting-substances (TBARS) assay, and the formation and attenuation of Reactive Oxygen Species (ROS) in RAW 264.7 macrophage cells. The results are being disseminated to communities of interest through the publication process and by presentation. First year results will be presented at the 30th Symposium on Biotechnology for Fuels and Chemicals, to be held in New Orleans in May, 2008.
PARTICIPANTS: The following people have been instrumental in planning and executing the tasks on this proposal: Dr. E.C. Clausen, PI, Professor, Chemical Engineering Dr. J.O. Lay, co-PI, Director, Arkansas Statewide Mass Spectrometry Facility Dr. S.N. Wallace, Research Associate, Chemical Engineering Dr. J.A. Gidden, Research Assistant, Chemistry/Biochemistry Dr. D.J. Carrier, Associate Professor, Biological Engineering Dr. W.R. Penney, Professor, Chemical Engineering Dr. Clausen is providing overall guidance and direction for the project, and Dr. Lay is coordinating the thermal degradation compound identification studies. Dr. Wallace is performing the degradation studies using batch reactors and the athero-protective effects studies, as measured by the TBARS assay, and the formation and attenuation of ROS in RAW 264.7 macrophage cells. Ms. Gidden is carrying out degradation compound identification using LC/MS, LC/MS/MS and exact mass determination. Dr. Carrier is providing guidance
and direction for the degradation and athero-protective effects studies. Finally, Dr. Penney is providing valuable expertise in the design of the countercurrent extractor for the second year of the study. This project offers ample opportunities for training or professional development including the training of post-doctoral research associates, graduate students and undergraduate Honors students. Dr. Wallace is currently receiving post-doctoral training by performing the degradation and athero-protective effects studies, and Ms. Gidden is receiving valuable hands-on experience in natural compound identification. As the project enters Year 2, an effort will be made to emphasize the training of undergraduate Honors students.
TARGET AUDIENCES: Subcritical water has received significant attention as an effective solvent for the recovery of active compounds from plant material because it is non-toxic, environmentally friendly, and is inexpensive. In ground-breaking work, this laboratory showed significant degradation of the extracted flavonolignans when extracting the fruits of S. marianum with subcritical water. This present effort seeks to identify, quantify, and then minimize the typical thermal degradation products formed during extraction. This information will be of significant value to industries and researchers seeking to utilize subcritical hot water for extraction.
PROJECT MODIFICATIONS: In addition to utilizing the TBARS assay to evaluate the athero-protective effects of extracted flavonolignans from S. marianum, the Reactive Oxygen Species (ROS) assay was added to the proposed work to measure oxidation in RAW 264.7 cells. This assay is a broad indicator of the antioxidant potential of a compound, and it also yields more information than the single electrophoretic mobility tests proposed in the original grant proposal.
Impacts
To generate thermal degradation products from the S. marianum flavonolignans, 5 mg of each of the individual flavonolignans (silichristin, silidianin, silibinin and isosilibinin) were first dissolved in 2 mL of methanol and added to 198 mL of deionized water. The individual solutions were then exposed to subcritical water at 500 kPa and 413 K for 0.5 hr. The agitation rate in the batch vessel was 150 rpm. Each 200 mL extract was dried down using a SpeedVac and reconstituted in methanol for HPLC-UV and LC/MS/MS analyses. Aliquots were reconstituted in DMSO and titrated to generate a dose response for subsequent assays. The extent of LDL oxidation was determined by TBARS measurement in a microplate assay. The chromatographic profiles of each of the four flavonolignans changed after exposure to subcritical water. HPLC-UV analysis showed silichristin appears as three isomers with distinct peaks eluting at 15.8, 17.4, and 18.9 min. After exposure to subcritical water, the
peak at 15.8 min disappeared and a new peak appeared at 7.4 min. Silibinin, which by HPLC-UV analysis appears as two diastereoisomers eluting at 24.6 and 25.7 min, retained the chromatographic profile of its diastereoisomers upon exposure to subcritical water, but gained two degradation products, which eluted at 18.4 and 26.3 min. Isosilibinin, when exposed to subcritical water, formed the most degradation products of all the tested flavonolignans. The diasteroisomers appeared both before and after exposure to subcritical water at approximately 28.0 and 29.0 min. Upon exposure to subcritical water, degradation peaks appeared at 15.9, 20.4, 22.4, 23.6, 24.6, and 30.2 min. The presence of degradation products in the flavonolignan solutions did not diminish the protective effects of silibinin, silidianin, or isosilbinin. Dose-dependent inhibitions in TBARS formation were observed in 0-300 μM silibinin, silydianin, and isosilibinin, which paralleled the inhibitions observed when
using the same doses of the compounds exposed to subcritical water. Silichristin displayed different protective effects before and after exposure to subcritical water. 0-300 μM silichristin resulted in decreasing TBARS levels with increasing doses of silichristin. After exposure to subcritical water, decreasing TBARS levels occurred with decreasing doses of post-exposure silichristin. The lowest tested dose of 9.4 μM showed the greatest degree of protection; the highest tested dose of 300 μM showed the least. The reversal in the trends exerted by silichristin could be explained by the complete disappearance of one of the silichristin isomers during exposure to subcritical water. These experiments show that clearly identifiable thermal degradation products are produced during the subcritical water extraction of S. marianum fruits. With the exception of silichristin, the presence of thermal degradation products does not diminish the protective effects of the
flavonolignans. The exposure of silichristin to subcritical water results in a diminished capacity for this flavonolignan to exert anti-atherosclerotic effects.
Publications
- Wallace, S.N., Raible, J., Carrier, D.J., Vaughn, K.L., Griffis, C.L., Clausen, E.C. and Nagarajan, S. 2007. Pressurized water rivals ethanol as a Silybum marianum extraction solvent for the inhibition of low-density lipoprotein oxidation mediated by copper and macrophage cells. Canadian Journal of Physiology and Pharmacology 85: 894-902.
|